Fluorescence is a process in which a molecule having suitable properties absorbs a photon of a given wavelength (the excitation process), resulting in the molecule in the excited state being raised to a higher energy level. The molecule referred to as a fluorophore can then drop down to a lower energy state by releasing a photon at a higher wavelength (lower energy), in the emission process. This energy can also be lost as heat to the surrounding environment, without the emission of a photon. The ratio of photons absorbed to emitted is known as the quantum efficiency. The difference between the excitation and emission spectral peak wavelengths is known as the Stokes shift.
The average time a fluorophore stays in the excited state prior to photon emission is known as its lifetime. This is an exponential decay process, following a rate defined by e−rt, where ┌ is the decay rate which is the inverse of the fluorescence lifetime and t is the time. Fluorescent probes are commercially available having a wide range of lifetimes, from less than 1 ns to well over 1 microsecond. All properties of a fluorescent probe are taken into consideration when making a choice for a particular application. Probes having longer lifetimes typically have lower quantum yields.
Another factor in the performance of fluorescent probes is their absorptivity, or ability to ‘capture’ photons. Probes having a high absorptivity and a high quantum yield can be considered as high performance. For a given photon flux rate molecules that absorb more photons will have more to emit, independent of quantum yield.
Covalent attachment of a fluorophore to an oligonucleotide primer can be used to label a DNA fragment. When different colored fluorophores are used for the bases A, C, G and T, then the fluorescence can be used to sequence DNA. Protein microarrays can be used to identify a myriad of biological players including protein—protein interactions, the ligands for receptors or binding proteins, the substrates of protein kinases, or the proteins that activate transcription factors. The array can be a substrate upon which capture probes have been affixed at separate locations with appropriate fluorophores in an ordered manner. Fluorescent detection is compatible with standard microarray scanners, the spots on the resulting image can be quantified by commonly used microarray quantification software packages.
Fluorescent proteins are commonly used in order to undertake live cell imaging. The fluorescent proteins can be highly specific bio sensors used to monitor a wide range of intracellular phenomena, including pH, metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. Flow cytometry can be used to count and examine cells by suspending them in a stream of fluid and passing them by fluorescence detection system. A wide range of fluorophores can be used as labels in flow cytometry. Fluorophores can be conjugated to a protein that recognizes a target feature on or in the cell. Different fluorophores with characteristic excitation and emission wavelengths can be used to label different features. In some cases, the emission wavelengths can overlap with the excitation wavelength of the same or another fluorophore.